Cancer cells differ from normal cells in a variety of ways, one of which is the molecular composition of the cell surface. These differences may be exploited in the development of targeted therapeutics. In principle, a variety of anti-neoplastic agents can be attached to affinity molecules, such as monoclonal antibodies (mAbs), which recognize tumor-specific cell surface molecules to achieve targeted killing (Carter (2001) Nature Rev Cancer, 1: 118-129). The epitope space at the tumor cell surface, however, is highly complex, including, in addition to proteins, carbohydrate determinants and other post-translational modification products which are difficult to probe by gene expression-based approaches (Theocharis (2002) Biochim Biophys Acta., 1588: 165-172; Lee et al. (2002) J Am Chem Soc, 124: 12439-12446). For carcinoma of the prostate (CaP), there are few known specific cell surface markers, and even fewer specific markers for hormone refractory CaP (Fair et al. (1997) Prostate. 32: 140-148; Dhanasekaran et al. (2001) Nature, 412: 822-826; Xu et al. (2000) Cancer Res., 60: 6568-6572; Hubert et al. (1999) Proc Natl Acad Sci USA., 96: 14523-14528; Saffran et al. (2001) Proc Natl Acad Sci USA., 98: 2658-2663; Reiter et al. (1998) Proc Natl Acad Sci USA., 95: 1735-1740). Moreover, despite recent advances in early diagnosis and treatment, prostate cancer remains the most common and second most lethal tumor in American men (Jemal et al. (2003) CA Cancer J Clin, 53: 5-26), and no curative treatment currently exists for metastatic disease (Coleman (2002) Am J Clin Oncol, 25: S32-38, 2002).
Tumor specific epitope space may be efficiently mapped by complementary mAbs. Phage display of non-immune single chain Fv (scFv) or Fab antibody repertoires has proven to be an important tool for generating highly specific antibody combining sites that may be readily converted into mAbs (Marks et al. (1991) J Mol Biol, 222: 581-597; de Haard et al. (1990) Nature, 348: 552-554; Barbas et al. (1991) Proc Natl Acad Sci USA, 88: 7978-7982), if needed. Non-immune phage libraries are derived from naïve human lymphocytes, thus recapitulate the primary immune response (Marks et al. (1991) J Mol Biol, 222: 581-597), and overcome difficulties with generating antibodies to evolutionarily conserved, or ‘self’, antigens which may comprise a large portion of tumor antigens (Sheets et al. (1998) Proc Natl Acad Sci USA, 95: 6157-6162; Griffiths et al. (1993) Embo J, 12: 725-734). This broader repertoire of specificities allows a less biased and more thorough mapping of epitope space (Amersdorfer et al. (1993) Vaccine, 20: 1640-1648). MAbs to tumor antigens have been isolated by directly selecting phage libraries on native and modified tumor antigens (Hoogenboom (2002) Methods Mol Biol, 178: 1-37). The success of direct cell selections in generating tumor targeting mAbs has been limited, however, by high non-specific binding of phage to cell surfaces and by high representation of phage antibody binding to common cell surface molecules (Hoogenboom (2002) Methods Mol Biol, 178: 1-37; Gao et al. (2003) J Immunol Methods, 274: 185-197).
Recently we reported that phage antibody selections on cells could be significantly improved by selecting for mAbs that trigger receptor mediated endocytosis, since endocytosed phage may be recovered from within the tumor cell after stripping non-specific binders from the cell surface (Becerril et al. (1999) Biochem Biophys Res Commun, 255: 386-393). Besides increasing selection efficiency, this approach generates mAbs that have desirable biologic properties: receptor mediated intracellular drug delivery, induction of apoptosis, or inhibition of proliferation (Nielsen et al. (2002) Biochim Biophys Acta., 1591: 109-118; Poul et al. (2000) J Mol Biol, 301: 1149-1161; Heitner et al. (2001) J Immunol Methods, 248: 17-30). We applied this approach to breast tumor cells and generated mAbs to a number of known internalizing receptors, including EGFR and ErbB2 (Poul et al. (2000) J Mol Biol, 301: 1149-1161; Heitner et al. (2001) J Immunol Methods, 248: 17-30). To broaden applicability, libraries of phage displaying multiple copies of scFv were engineered (O'Connell et al. (2002) J Mol Biol, 321: 49-56; Liu and Marks (2000) Anal Biochem, 286: 119-128; Huie et al. (2001) Proc Natl Acad Sci USA., 98: 2682-2687), which unlike existing phage libraries can crosslink receptors, allowing more efficient phage endocytosis (Becerril et al. (1999) Biochem Biophys Res Commun, 255: 386-393).